Project Summary Saliva performs a number of extremely important biological functions that are instrumental in maintaining oral health. It has been estimated that more than 5 million people in the US suffers from salivary gland dysfunctions that occurs as a results of various pathological conditions such as radiation therapy for head and neck cancer and Sjgerns syndrome. Loss of saliva in these patients causes mastication and swallowing difficulties, burning sensation to the mouth and dysgeusia. Although current treatment options reduce the severity of the symptoms, they cannot restore acini function. Thus, efforts to restore salivary gland tissues are of utmost importance for these patients. Importantly, stem cells have been suggested as possible source for tissue regeneration; it has not been used for salivary gland regeneration. One of the major issues in using stem cells for regeneration is that soon after introduction of these stem cells in an organ system, they start differentiating, thereby not providing enough stem cells that can restore the organ function. Results obtained from our ongoing studies indicate that loss of calcium entry induces stem cell differentiation, thereby inhibiting stem cell proliferation. Furthermore we have identified that Ca2+ entry in mesenchymal stem cells is mediated via the store-operated Ca2+ entry (SOCE) mechanism that is dependent on TRPC1-STIM1 interaction. Importantly, overexpression of a mutant STIM1 construct (STIM1D76A) functionally interacts with TRPC1-Orai1 complex (plasma membrane Ca2+ channels that function as SOCE channels) and constitutively activates Ca2+ entry that is essential for cell proliferation. These results are novel, and suggest a strong relationship with regard to changes in calcium handling that can promote proliferation, but they need to be further validated. Therefore, in this grant proposal we intend to thoroughly characterize the role of changes in calcium signaling and to determine if manipulation of these calcium signaling modulators can restore radiation induced damaged salivary glands. The hypothesis of this study is that regulated Ca2+ entry can support c-kit positive mesenchymal stem cell proliferation that can regenerate salivary gland cells and restore salivary secretion. Thus, identification of the mechanism as well as the factors that lead to salivary gland restoration could represent as new therapeutic options for patients that suffer from salivary gland dysfunction. We have initiated collaboration with a group, who have developed this technique and will coordinate our efforts in order to determine the functional significance of calcium channel in regulating stem cell proliferation and differentiation to restore saliva secretion. We will coordinate our efforts in order to determine the functional significance of mesenchymal stem cells in restoring salivary gland function. We will also investigate the mechanism and the ion channels involved in stem cell proliferation/differentiation. The results of our studies are expected to provide new insights into the role of stem cells as possible reagents for salivary gland regeneration. Greater understanding of these events will be important in elucidating new therapy for patients that suffer from salivary gland dysfunctions.